Our efforts to understand the structures of molecules relevant to immune recognition have, in the past year, focused on three prototype molecules: MHC class I molecules, natural killer (NK) receptors, and T cell receptors. These efforts are based on our laboratory?s ability to engineer, refold, purify and characterize in various binding studies the relevant molecules. We have been particularly interested in the MHC-I molecule, H-2Dd, which is of unique interest in that it not only binds specific T cell receptors, but it also binds the murine natural killer cell receptor, Ly49A. We have engineered soluble versions of H- 2Dd and a TCR that binds H-2Dd/P18-I10 complexes, expressed these proteins in bacteria and refolded and purified them to homogeneity from solubilized inclusion bodies. The interactions between these molecules have been examined by surface plasmon resonance and by analytical equilibrium ultracentrifugation. In collaboration with Dr. Roy Mariuzza?s group at University of Maryland, we have determined the crystal structure of the H-2Dd/P18-I10 complex to 3.1 Angstroms resolution. The bound peptide is in an unusual backbone configuration. With respect to TCR structure, we have completed the high resolution structural analysis of a TCR Valpha domain by both x-ray crystallography (in collaboration with Dr. E. Padlan, NIDDK) and by high resolution nuclear magnetic resonance (NMR) (in collaboration with Drs. J.-S. Shan and A. Bax, NIDDK). With final refinement of the three dimensional structure by NMR, this will be compared to that of the Valpha in the crystal. Further studies allowing assignment of the specific residues involved in binding to the peptide/MHC complex in solution will be evaluated. Our most exciting progress in the past year has been our success in obtaining crystals of the mouse NK receptor, Ly49A complexed with H-2Dd. (This has been a vigorous collaboration between our laboratory (K. Natarajan) and that of R. Mariuzza (J. Tormo), in which protein expression, purification, and characterization have been carried out by Dr. Natarajan, and crystallization and data collection by Dr. Tormo). We engineered in bacteria a fragment that crystallized (Ly49A T) that began at residue 127, which we then engineered for bacterial expression. Ly49A T retained the ability to bind H-2Dd, and crystals of it complexed with H-2Dd/P18-I10/beta2m were grown at 4?C in hanging drops. Crystals were washed and shown to contain both H-2Dd and Ly49A molecules in a molar ratio of 1:2 by SDS- PAGE. Crystallographic data were collected from cryopreserved crystals. Preliminary characterization of the data set indicates that the crystals belong to the space group P21212 with unit cell dimensions: a= 78.96 Angstroms , b = 96.96 Angstroms, c = 99.32 Angstroms, and contain one H-2Dd molecules and one Ly49A dimer per asymmetric unit. Data were collected to 2.3 Angstroms representing 94.7% completeness overall, and 80.7% completeness in the 2.35 to 2.3 Angstrom resolution shell. Structure determination, using molecular replacement methods to obtain phase information, is presently underway. - NK receptor, T cell receptor, X-ray crystallography